Natural killer cell (NK) receptors for major histocompatibility complex (MHC) class I influence engraftment and graft-versus-tumor effects after allogeneic bone marrow transplantation. We find that SH2-containing inositol phosphatase (SHIP) influences the repertoire of NK receptors. In adult SHIP-/- mice, the NK compartment is dominated by cells that express two inhibitory receptors capable of binding either self or allogeneic MHC ligands. This promiscuous repertoire has significant functional consequences, because SHIP-/- mice fail to reject fully mismatched allogeneic marrow grafts and show enhanced survival after such transplants. Thus, SHIP plays an important role in two processes that limit the success of allogeneic marrow transplantation: graft rejection and graft-versus-host disease.
Mutations in chs1/beige result in a deficiency in intracellular transport of vesicles that leads to a generalized immunodeficiency in mice and humans. The function of NK cells, CTL, and granulocytes is impaired by these mutations, indicating that polarized trafficking of vesicles is controlled by CHS1/beige proteins. However, a molecular explanation for this defect has not been identified. Here we describe a novel gene with orthologues in mice, humans, and flies that contains key features of both chs1/beige and A kinase anchor genes. We designate this novel gene lba for LPS-responsive, beige-like anchor gene. Expression of lba is induced after LPS stimulation of B cells and macrophages. In addition, lba is expressed in many other tissues in the body and has three distinct mRNA isoforms that are differentially expressed in various tissues. Strikingly, LBA-green-fluorescent protein (GFP) fusion proteins are localized to vesicles after LPS stimulation. Confocal microscopy indicates this protein is colocalized with the trans-Golgi complex and some lysosomes. Further analysis by immunoelectron microscopy demonstrates that LBA-GFP fusion protein can localize to endoplasmic reticulum, plasma membrane, and endocytosis vesicles in addition to the trans-Golgi complex and lysosomes. We hypothesize that LBA/CHS1/BG proteins function in polarized vesicle trafficking by guiding intracellular vesicles to activated receptor complexes and thus facilitate polarized secretion and/or membrane deposition of immune effector molecules.
IntroductionEngagement of receptors on the surface of mammalian cells results in the activation of phosphatidylinositol 3-kinase (PI3K) and phosphorylation of phosphatidylinositols on the cytoplasmic side of the cell membrane. 8,15 The generation of phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5 P 3 ) by PI3K contributes to the activation of signaling pathways that drive cell proliferation. [4][5][6][7] Removal of the phosphate group from the D5 position of phosphatidylinositols by the SH2-containing inositol 5Ј-phosphatase (SHIP) is now recognized as an important negative feedback mechanism on cell activation in the mammalian hematopoietic compartment. 8,9 SHIP was identified based on its ability to bind Grb2, 10 Shc,11,12 and the Fc␥RIIB receptor 13 and by gene trapping. 14 In vitro assays show that SHIP can remove the 5Ј-phosphate of PI 3,4,5 P 3 or inositol 1,3,4,5-tetrakisphosphate, 10,11 suggesting that SHIP may counteract the activity of PI3K 8,9,15 or prevent the sustained influx of Ca ϩϩ into the cell. 9,16,17 SHIP can also form a complex with Shc in stimulated cells. [17][18][19][20][21] Formation of a SHIP-Shc complex is proposed to prevent the recruitment of the nucleotide exchange factor mSos1 to the membrane, thus preventing the activation of the Ras/mitogenactivated protein kinase (MAPK) pathway. 21,22 The ability of SHIP to convert PI 3,4,5 P 3 to PI 3,4 P 2 suggests that SHIP might also influence the activation of protein kinase B/Akt and thus influence the induction of programmed cell death. 7,23,24 There is increasing genetic evidence that SHIP plays an important role as a negative regulator of cell activation in B-lymphoid cells, [1][2][3]6,16 myeloid cells, 1,3 and mast cells. 25 These genetic studies and biochemical analysis of SHIP phosphorylation status indicate that SHIP responds to a wide variety of signals in the hematopoietic compartment, including cytokines, 18,20,26 antigen, 17,19,20 immune complexes, 13,16,27,28 the Fc portion of immunoglobulin G (IgG) antibodies, 29 and thrombin. 30 These results demonstrate that SHIP is an important regulator of cellular responses in the mature cells of several hematopoietic lineages.Here we describe the cloning and characterization of a novel SHIP isoform, s-SHIP. We show that transcription from an internal site within the SHIP gene promotes the expression of s-SHIP in totipotent embryonic stem (ES) cells and hematopoietic stem cells (HSCs), but not in mature hematopoietic cells. The s-SHIP isoform lacks the SH2 domain found in the previously described SHIP isoform whose expression is restricted to the hematopoietic system. Consistent with this structural difference, s-SHIP does not associate in vivo with the Shc adapter protein, but it does associate with Grb2. We propose that s-SHIP plays a unique signaling role in primitive stem cell populations. Materials and methods Cell cultureThe ES cell line, TL1, was a kind gift of Dr Patricia A. Labosky 31 (University of Pennsylvania, Philadelphia) and was cultured in Dulbecco modified Eagle mediu...
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